Lorena Lopes Ferreira

Repellent activity of plant-derived compounds against Amblyomma cajennense (Acari: Ixodidae) nymphs

Repellence responses of Amblyomma cajennense nymphs to callicarpenal, intermedeol, Hyptis suaveolens essential oil, extract of Melia azedarach, Cymbopogon nardus, Spiranthera odoratissima, Chenopodium ambrosioides, Ageratum conyzoides, Mentha pulegium, Ruta graveolens, and Memora nodosa were studied. Among these the extract of C. nardus stood out because of the long-lasting repellence, maintaining, in the highest concentration, 35h of protection against 90% of the nymphs. The essential oil of H. suaveolens and the extracts of C. ambrosioides and A. conyzoides showed good repellence index (66%) when applied in high concentrations. However, greater protection could be obtained at higher concentrations but with a shorter repellence time. Callicarpenal, intermedeol, extract of M. Pulegium, and M. nodosa leaves showed moderate repellence in high concentrations. Extracts from M. azedarach, R. graveolens, S. odoratissima, and M. nodosa roots showed little or no repellent effect. These results show that some plant extracts may represent a promising alternative in the control of infestations by A. cajennense.

Identification of non-host semiochemicals for the brown dog tick, Rhipicephalus sanguineus sensu lato (Acari: Ixodidae), from tick-resistant beagles, Canis lupus familiaris

Studies have shown that the brown dog tick, Rhipicephalus sanguineus sensu lato, when fed on the beagle breed of dog, Canis lupus familiaris, development negatively affected in comparison with tick development after feeding on the English cocker spaniel breed. Thus leading to the suggestion that beagle dogs are be tick-resistant dogs. Behavioural studies have demonstrated that more ticks are attracted by extracts from cocker spaniels than from beagles and that the odour of beagles is a repellent. To test the hypothesis that resistant hosts produce repellent compounds, we undertook comparative chemical analysis on beagle odour and cocker spaniel extracts using coupled high-resolution gas chromatography-mass spectrometry (GC-MS) and also used Petri-dish and olfactometer behavioural assays to assess the response of ticks to identified non-host compounds. The beagle odour extracts contained almost three times as many chemical compounds as cocker spaniel samples. Several non-host compounds were identified, i.e. 2-hexanone, benzaldehyde, nonane, decane and undecane. In Petri-dish assays, 2-hexanone was repellent at 30 min at concentrations of 0.200 and 0.050 mg cm(-2), whilst at 10 min, the 0.100 mg cm(-2) concentration was repellent. Benzaldehyde repelled ticks at 30 min (0.200 mg cm(-2)) and at 5 min (0.050 mg cm(-2)). Undecane was repellent for R. sanguineus s.l. ticks for the first 5 min at the highest concentration tested. Nonane and decane did not show any significant repellency at any concentration or time evaluated. When 2-hexanone and benzaldehyde were combined, an increase in the repellency rate was observed, with activity comparable or better than N,N-diethyl-3-methylbenzamide (DEET). In olfactometer bioassays, a 1:1 mixture of 2-hexanone:benzaldehyde and DEET were repellent for R. sanguineus s.l. adults at the concentration of 0.200 mg cm(-2). This study identified non-host semiochemicals that mediate avoidance of the beagle dog breed by R. sanguineus s.l. This finding may enable development of new approaches to control this tick.

Role of Rhipicephalus microplus cheliceral receptors in gustation and host differentiation.

Rhipicephalus microplus is considered the most economically important ectoparasite of cattle worldwide. It is known that zebuine breeds of cattle are less susceptible to tick infestation than taurine breeds. Contact chemoreceptors in the cheliceral pit sensilla of ticks respond selectively to phagostimulant compounds, however their role in blood feeding relative to host susceptibility to infestation remains to be fully understood. We addressed this topic by conducting taste electrophysiology experiments with cheliceral pit sensilla preparations of R. microplus females. Solutions of five known ixodid tick phagostimulants were tested at different concentrations: sodium (NaCl), and potassium chloride (KCl) (10(-3)-10(-1)M); glucose (10(-4)-10(-1)M); adenosine triphosphate (ATP) (10(-6)-10(-2)M); and reduced l-glutathione (GSH) (10(-6)-10(-2)M). Serum samples from six susceptible animals of the Girolando breed (5/8 Bos indicus×3/8 B. taurus) and six resistant Nelore bovines (pure B. indicus) were also tested. A dose-dependent response of gustatory neurons associated with the chelicerae sensillum to NaCl, glucose, GSH, and ATP were observed. Responses by the cheliceral inner digit pit sensilla of R. microplus to KCl and glucose were also observed and they are reported here for the first time. In addition to an electrophysiological response to known phagostimulants, chemoreceptors in the chelicera of R. microplus responded differently to serum from cattle susceptible and resistant to infestation. The cheliceral pit neurons were more responsive to serum of R. microplus resistant bovines with a higher mean spike frequency (53.5±2spikess(-1)) than to serum samples from susceptible cattle (40.3±2spikess(-1)). The implications of chemosensation during tick blood feeding are discussed.

In vitro repellency of DEET and β-citronellol against the ticks Rhipicephalus sanguineus sensu lato and Amblyomma sculptum

Rhipicephalus sanguineus sensu lato and Amblyomma sculptum can parasite humans and domestic animals and are vectors of pathogens, including zoonoses. Repellents are an important tool of tick control. This study aimed to compare the efficacy of N,N-diethyl- 3-methylbenzamide (DEET), a standard repellent, versus β-citronellol in a Petri dish bioassay. A semicircle of filter paper (31.8cm2) was treated with 87μl of one of four concentrations (0.200, 0.100, 0.050 and 0.025mg/cm2) of β-citronellol, DEET or solvent (ethanol). A head-to-head test was developed treating one side with increasing concentrations of β-citronellol as above mentioned, against the highest concentration of DEET. Besides that a blank assay was performed. Three males and three females were placed in the middle of the plate and their location was evaluated 5, 10 and 30min after the test was initiated. As a result, the time had no significant effect on repellency response of the ticks exposed to both compounds and their concentrations. The repellency response raised according with the increase of concentration. Additionally, our findings indicate that the tick A. sculptum was more sensitive to the compounds tested and β-citronellol showed a higher efficacy than DEET. In addition, β-citronellol could be formulated to protect humans and other animals from R. sanguineus s. l. and A. sculptum infestation, as well as the diseases transmitted by these species.

Quantification of brown dog tick repellents, 2-hexanone and benzaldehyde, and release from tick-resistant beagles, Canis lupus familiaris

We have recently shown that repellency of the tick Rhipicephalus sanguineus sensu lato by the tick resistant dog breed, the beagle, is mediated by volatile organic compounds (VOCs) 2-hexanone and benzaldehyde present in beagle odour. Ectoparasite location of animal hosts is affected by variation in these odour components and their ratios. The aim of this study was to quantify the release rate, and the ratio, of 2-hexanone and benzaldehyde from beagles. The odour of three beagles was collected, for four days, over one week (day 0, day 1, day 4 and day 7). The compounds were identified using coupled high-resolution gas chromatography-mass spectrometry (GC-MS), and authentic standards of compounds were used to generate external calibration curves for quantification. Both compounds were found in all dogs on all days. The amount of benzaldehyde was always higher than that of 2-hexanone and so their ratio varied from unity, on average (over time) being 3.128±0.365, 1.902±0.390, 1.670±0.671ngmL(-1) for beagle 1, 2 and 3, respectively. There was no significant (p<0.05, F-test) effect of time. The overall mean was 2.233±0.387ngmL(-1). These results further previous findings by documenting the presence of 2-hexanone and benzaldehyde in beagle odour samples covering a 7-day period. This knowledge enables development of repellents to protect dogs from R. sanguineus s. l. infestation.

Induction of complete courtship ritual in Amblyomma cajennense using 2,6-dichlorophenol at female-equivalent quantities

In order to clarify the role of 2,6-dichlorophenol (2,6-DCP) in the courtship of Amblyomma cajennense, sexually mature males that had previously fed on rabbits were tested in bioassays. The males were released onto dummies treated with whole female extract or synthetic 2,6-DCP at a concentration of two female equivalents, or with hexane (control), and their responses were observed. In the presence of both the extract and 2,6-DCP, excitation was observed among the males, expressed in the form of touching and probing the dummy, and mounting occurred readily. The percentages of mounting (73%) and tipping over (60%) were equal in the two treatments and higher than in the control group (27 and 20%, respectively). Relatively short durations of mounting were recorded, and these were statistically similar in all treatments. Almost all instances of mounting resulted in tipping-over behavior. A few isolated cases of males that went directly to ventral positioning without mounting were observed. It was confirmed that 2,6-DCP alone is capable of mediation of mounting behavior in A. cajennense.

Comparison of two methods to quantify 2,6-dichlorophenol from tick Amblyomma cajennense by GC/MS-SIM

Amblyomma cajennense (Acari: Ixodidae) e um carrapato de grande importância socioeconomica no subcontinente Sul-Americano. Apesar disso, pouco se conhece acerca de sua ecologia quimica, cuja informacao e crucial para o seu controle. Nesse estudo, 2,6-diclorofenol (2,6-DCP), o feromonio sexual de A. cajennense foi quantificado por cromatografia gasosa acoplada a espectrometria de massas no modo de analise por monitoramento seletivo de ions (CG/EM-MSI) a partir de femeas alimentadas em coelhos durante 6 dias. O extrato do feromonio sexual foi obtido pela exposicao ao ultrassom de femeas virgens em hexano em duas amostras independentes. Nenhum pre-tratamento da amostra foi necessario. Os metodos de adicao padrao (SA) e de curva de calibracao com 5-bromo-4-hidroxi-3-metoxibenzaldeido (5-BrV) como padrao interno (IS) foram utilizados para a quantificacao. O conteudo de 2,6-DCP nao apresentou diferenca significativa entre os extratos e/ou metodos utilizados. Os resultados mostraram que as faixas de concentracao de 2,6-DCP por femea foram de 2,03-2,27 ng mL -1 e de 2,06-2,24 ng mL -1 para os metodos SA e IS, respectivamente. Os metodos mostraram ser especificos, sensiveis e fidedignos na determinacao de 2,6-DCP em carrapatos.

Brown dog tick, Rhipicephalus sanguineus sensu lato, infestation of susceptible dog hosts is reduced by slow release of semiochemicals from a less susceptible host

Domestic dog breeds are hosts for the brown dog tick, Rhipicephalus sanguineus sensu lato, but infestation levels vary among breeds. Beagles are less susceptible to tick infestations than English cocker spaniels due to enhanced production of 2-hexanone and benzaldehyde that act as volatile tick repellents. We report the use of prototype slow-release formulations of these compounds to reduce the burden of R. sanguineus s. l. on English cocker spaniel dogs. Twelve dogs were randomly assigned to two groups with six dogs each. The treated group received collars with slow-release formulations of the compounds attached, while the control group received collars with clean formulations attached. Five environmental infestations were performed, with the number of ticks (at all stages) on the dogs being counted twice a day for 45days. The counts on the number of tick stages found per dog were individually fitted to linear mixed effects models with repeated measures and normal distribution for errors. The mean tick infestation in the treated group was significantly lower than in the control group. For larvae and nymphs, a decrease in tick infestation was observed at the fifth count, and for adults, lower average counts were observed in all counts. The compounds did not interfere with the distribution of the ticks on the body of the dogs, as a similar percentage of ticks was found on the anterior half of the dogs (54.5% for the control group and 56.2% for the treated group). The biological and reproductive parameters of the ticks were not affected by the repellents. This study highlights for the first time the potential use of a novel allomone (repellent)-based formulation for reduction of tick infestation on susceptible dogs.

Chemical composition and biological characterization of extracts of male-produced pheromone from Amblyomma cajennense (Fabricius, 1787) (Acari: Ixodidae) by GC/MS-SIM and behavioural responses

The extract from fed Amblyomma cajennense (Fabricius) males was characterized for chemical composition and behavioural responses. The pheromone was obtained by extraction in hexane, analysed by gas chromatography/mass spectrometry-single ion monitoring and subjected to behavioural bioassays in vitro and in vivo. The presence of 10 constituents already identified in tick pheromones was evaluated and qualitative analysis of the extract indicated the presence of benzaldehyde, benzoic acid, nonanoic acid, salicylic acid, 2,6-dichlorophenol, limonene and methyl salicylate and the absence of butyric acid, isobutyric acid and o-nitrophenol. In an olfactometer bioassay, males, females and nymphs were not attracted by the extract (P > 0.05) as the displacement angles (0°–10°) distribution was similar between tests (13.3–38.6%) and controls (8.5–23.7%). Similarly, there was no statistically significant aggregation response of test ticks to extracts in bioassays in vitro. However, significantly greater attachment (P < 0.05) in tests with extracts was registered within a 24-h period than that in the control in males but not in females. A synthetic simulation, constituted according to the proportions obtained in the chemical analysis, yielded comparable results. The behavioural observations made during the study were consistent with the extracts chemical composition. The potential implication of the findings in the management of A. cajennense in the regions where it is endemic is discussed.

Cyrtoneurina pararescita (Diptera: Muscidae) (Couri, 1995) como novo hospedeiro de Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae)

This study reports the occurrence of Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) as parasitoid of Cyrtoneurina pararescita (Diptera: Muscidae) (Couri, 1995) in bovine feces obtained in Goiânia, Goias state, Brazil. Bovine manure samples, collected at two weeks intervals, were taken to the laboratory and the pupae were extracted by water flotation. Each pupa was placed in capsules of gelatin until the emergence of dipterous or their parasitoids. The parasitism percentage was of 2.4%. This paper reports the first occurrence of N. vitripennis as parasitoid of C. pararescita worldwide.